The present invention relates generally to a method of identifying modulators of biological interactions and agents useful for same. More particularly, the present invention contemplates a method of detecting inhibitors of biological interactions involving proteinaceous and/or nucleic acid molecules and more particularly a method of identifying peptide inhibitors of biological interactions having adverse effects on living cells, tissue or organisms. The present invention provides the means by which a wide range of peptide-based therapeutic, prophylactic and diagnostic reagents may be developed.
This specification contains nucleotide and amino acid sequence information prepared using the programme PatentIn Version 2.0, presented herein after the bibliography. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator  less than 210 greater than  followed by the sequence identifier (e.g.  less than 210 greater than 1,  less than 210 greater than 2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields  less than 211 greater than ,  less than 212 greater than  and  less than 213 greater than , respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field  less than 400 greater than  followed by the sequence identifier (eg.  less than 400 greater than 1 (SEQ ID NO: 1),  less than 400 greater than 2 (SEQ ID NO: 2), etc).
Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.
As used herein the term xe2x80x9cderived fromxe2x80x9d shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
Biological interactions, such as protein:protein interactions, protein:nucleic interactions, protein:ligand interactions and nucleic acid:nucleic acid interactions are involved in a wide variety of processes occurring in living cells. For example, agonism and antagonism of receptors by specific ligands, including drugs, hormones, second messenger molecules, etc. may effect a variety of biological processes such as gene expression, cellular differentiation and growth, enzyme activity, metabolite flow and metabolite partitioning between cellular compartments, amongst others. DNA:protein and RNA:protein interactions are well known for their effects in regulating gene expression in both prokaryotic and eukaryotic cells, in addition to being critical for DNA replication and in the case of certain viruses, RNA replication.
Undesirable or inappropriate gene expression and/or cellular differentiation, cellular growth and metabolism may also be attributable, at least in many cases, to biological interactions involving the binding and/or activity of proteinaceous molecules, such as transcription factors, peptide hormones, receptor molecules and enzymes, amongst others.
In one example, it is known that several genes activated by chromosomal translocations in lymphoid malignancies code for transcription factors, for example MYC, LYL-1 and SCL, which appear to function via protein-protein interactions. In normal cells, these proteins are in an appropriate equilibrium with their interaction partners which is disturbed as a consequence of oncogene activation and is thought to result in transcription of target genes normally expressed in other cells or lineages. These transcription factors may also substitute for, or antagonise, the function of closely related endogenous proteins to perturb gene expression essential for normal growth control.
Peptides present potential therapeutic and prophylactic agents for many human and animal diseases, biochemical disorders and adverse drug effects, because they can interact with other molecules highly specifically. For example, mimetic peptides have been reported to inhibit protein interactions and/or enzymic functions. More specific examples include a nonapeptide derived from the ribonucleotide reductase of herpes simplex virus which is linked to an enterotoxin subunit for delivery into cells via its receptor. The peptide conjugate is found to inhibit herpes simplex type I replication in quiescent Vero cells (Marcello et al. 1994). Using detailed knowledge of the PCNA-interaction domain of p21WAF1, a peptide was designed which effectively blocked the interaction. This 20-mer peptide bound with sufficient affinity to block SV40 replication (Warbrick et al. 1996). A 20-mer peptide sequence derived from p16 was found to interact with cdk4 and cdk6 and inhibited pRB phosphorylation and cell cycle progression (Fahraeus 1996). Peptides have even been shown to function as inhibitors in animal models. For examples, a peptide targeting the ICE protease was shown to be a potent protective inhibitor against liver apoptosis induced by TNF-xcex1 in the mouse (Rouquet et al. 1996).
A major problem to be overcome in the field of peptide therapeutics and prophylactics is the identification of specific amino acid sequences having a desired antagonist or agonist activity against a particular biological interaction in a particular cellular environment.
Additionally, in view of the wide range of possible applications of peptide therapeutics, the potential number of useful amino acid sequences is enormous. This poses a particular problem in terms of identifying, from the vast pool of potential amino acid sequences having utility, those amino acid sequences which have a specific activity under a particular set of cellular conditions.
There is currently no available method for screening random peptide libraries in vivo for the purpose of identifying specific peptides which inhibit specific protein interactions.
Accordingly, there is a need to develop technologies which provide for the rapid, large-scale determination of useful peptide therapeutics.
One aspect of the present invention provides a method of identifying a peptide, oligopeptide or polypeptide which is capable of modulating a biological interaction in a host cell said method comprising the steps of:
(i) producing a peptide library in a cellular host wherein the transformed cells of said library contain at least a first nucleotide sequence which comprises or encodes a reporter molecule the expression of which is operably under control of said biological interaction and a second nucleotide sequence which encodes said peptide, oligopeptide or polypeptide placed operably under the control of a suitable promoter sequence;
(ii) culturing said cellular host for a time and under conditions sufficient for expression of said second nucleotide sequence to occur; and
(iii) selecting cells wherein expression of said reporter molecule is modified.
A second aspect of the present invention contemplates peptides, oligopeptides and polypeptides identified by the method of the present invention.
Another aspect of the present invention contemplates a pharmaceutical composition comprising a peptide, oligopeptide and polypeptide which is capable of modulating a biological interaction and one or more pharmaceutically acceptable carriers and/or diluents.
Another aspect of the present invention provides a shuttle vector which is capable of expressing a first amino acid sequence as a fusion with a second amino acid sequence in which it is conformationally constrained, wherein said shuttle vector at least comprises:
(i) a first expression cassette comprising:
(a) a multiple cloning site for insertion of a first nucleotide sequence encoding said first amino acid sequence, wherein said multiple cloning site is adjacent to one or more second nucleotide sequences encoding a nuclear localisation motif and/or polypeptide loop such that a fusion polypeptide is capable of being produced between said first and second amino acid sequences;
(b) two or more tandem promoter sequences to which said first and second nucleotide sequences are operably connected in use wherein one of said promoter sequences is a bacterially-expressible promoter and wherein one of said promoter sequences is a yeast-expressible promoter; and
(c) a terminator sequence adjacent to the multiple cloning site and distal to said promoter sequence and second nucleotide sequences;
(ii) a bacterial origin of replication; and
(iii) a eukaryotic origin of replication.
In an alternative embodiment, the shuttle vector of the invention further comprises a second expression cassette comprising a selectable marker gene operably linked to two or more promoter sequences and placed upstream of a terminator sequence, wherein one of said promoter sequences is a bacterially-expressible promoter and wherein one of said promoter sequences is a yeast-expressible promoter.
In an alternative embodiment, the subject shuttle vector is further modified to provide for expression in mammalian cells, by introducing into the first and second expression cassettes mammalian cell-expressible promoter and terminator sequences in tandem array with the promoter and terminator sequences already present in the subject expression cassettes.
A further aspect of the invention provides a method of identifying an antagonist of a biological interaction in a bacterial cell, said method comprising:
(i) placing the expression of a reporter molecule operably under the control of a biological interaction in said cell, wherein at least one partner of said biological interaction comprises a peptide, oligopeptide, polypeptide or protein encoded by a nucleotide sequence that is placed operably in is connection with a bacterial-expressible promoter in pBLOCK-1 or a derivative thereof;
(ii) incubating the cell in the presence of a candidate compound to be tested for the ability to antagonise the biological interaction; and
(iii) selecting cells wherein expression of said reporter molecule is modified.